The treatment of haemorrhagic problems in haemophilia and other related disorders, such as liver disease, is carried out by means of substitution therapy with clotting factors.
The treatment of choice in haemophilia A (deficiency of clotting factor VIII) comprises the administration of said factor VIII (FVIII), both at the prophylactic level and in acute episodes. Unfortunately, one of the problems of therapy with FVIII is the appearance of FVIII-inhibiting antibodies, which reduces the efficacy of this treatment.
Other alternative treatments include the administration of concentrates of activated prothrombin complex (APCC). APCC consists of a mixture of vitamin K-dependent clotting factors (factors II, VII, IX and X) and other accompanying proteins (basically protein C and protein S). Treatment with these APCCs produces an increase in levels of the non-deficient factors, some of the factors moreover being found in their activated form, for which reason they can trigger thrombogenic phenomena in the patient, such as disseminated intravascular clotting.
The capacity of activated factor VII (FVIIa) for initiating clotting independently of the activity of factor VIII is of great therapeutic use, since it makes it possible to restore haemostasis in haemophilic patients who have developed antibodies that inhibit factor VIII and who, therefore, do not respond adequately to the substitution therapy. In comparison with other factors, FVIIa has a short biological half-life (about 4 hours), while the other factors of APCC have a longer half-life, which causes their accumulation.
Another advantage of treatment with purified FVIIa is that its action is localised by the availability of its co-factor, the tissue factor, which is released at the points of lesion (haemarthrosis, dental extractions, surgical interventions, etc.). At present, FVIIa tends to be considered as a global haemostatic agent, with a wide range of indications, such as overdose of dicumarins, liver failure, and unstoppable bleeding, among others.
In addition, in the administration of high purity FVIIa, through its high activity, the infusion of protein is minimal and other unnecessary proteins are not infused.
The preparations of FVIIa of plasmic origin have until now exhibited a relatively low FVIIa activity, in comparison with that of the present invention. Patent U.S. Pat. No. 4,479,938 for “Therapeutic composition containing factor VIIa” of BAXTER TRAVENOL LAB shows a low purity FVIIa, like patent EP0346241 for “Process for preparing a factor VIIa fraction, and its use as a medicament” of FOND NAT TRANSFUSION SANGUINE, which also shows the preparation of a low purity FVII, having an activity of between 95 and 130 IU of FVIIa/mg of protein.
The known methods of purification of FVIIa on an industrial level start from prothrombin complex or from equivalent fractions of plasmic fractioning, as shown by Patent EP0346241 and are based on methods which include selective precipitations and the use of centrifuging as a method for separating precipitates. As has been observed, in the purification processes based on precipitation, the degree of purification obtained is limited, describing moreover complex processes in which the separation of precipitates is by centrifuging, which is an industrially complex and expensive method.
Patent EP 0391974 for “Process for the purification of vitamin K-dependent blood clotting factors” of CENTRAL BLOOD LAB AUTHORITY refers basically to the purification of FIX, starting from prothrombin complex, by means of chromatography with a metal chelation column. As a by-product, proceeding from the washings from obtaining FIX, the procurement of FVII is claimed, not of FVIIa. The FVII obtained is of low purity (around one IU per mg of protein) and is characterized in that it does not contain FII, but may contain significant amounts of other vitamin K-dependent factors. By the method described, it is not possible to obtain a high purity FVIIa. Chromatography in a metal chelation column uses an activated resin with copper. In the method described in the present invention, the metal chelation chromatography uses a resin with nickel, which does not require prior activation; the washings are carried out at high conductivities, which permits stabilisation of the FVII in the column and elution does not require the presence of amino acids. This, linked also to the fact that the point of departure is a material of greater purity, makes it possible to obtain a product of high purity, which would not be achievable according to Patent EP 0391974.
Other methods for purification of plasmic FVIIa are based on the isolation and separation of FVII by immuno-affinity (with monoclonal antibodies) [Vox Sanguinis (2003) 84, 54-64]. With this method a high purity is obtained (specific activity of the order of 40,000 IU/mg of protein), but it is not possible to dismiss the presence, in the final product, of proteins of non-human origin coming from the monoclonal antibodies released by the resin used. These non-human proteins would be responsible for antigenic reactions in the patients treated.
Another aspect to be highlighted is the starting material for the process of purification of the FVIIa, which has conventionally been prothrombin complex (PTC) or an equivalent fraction. This implies that this material may be intended solely for obtaining one of the two products, either PTC or FVIIa. The possibility of purifying FVIIa starting from an alternative material, which may be a waste fraction not used in fractioning, such as the precipitate of the suspension of FrII+III, opens up the possibility of more efficient utilisation of the fractioned plasma.